Generation of antifungal effector CD8+ T cells in the absence of CD4+ T cells during Cryptococcus neoformans infection

Immunity to the opportunistic fungus Cryptococcus neoformans is dependent on cell-mediated immunity. Individuals with defects in cellular immunity, CD4(+) T cells in particular, are susceptible to infection with this pathogen. In host defense against a number of pathogens, CD8(+) T cell responses are dependent upon CD4(+) T cell help. The goal of these studies was to determine whether CD4(+) T cells are required for the generation of antifungal CD8(+) T cell effectors during pulmonary C. neoformans infection. Using a murine intratracheal infection model, our results demonstrated that CD4(+) T cells were not required for the expansion and trafficking of CD8(+) T cells to the site of infection. CD4(+) T cells were also not required for the generation of IFN-gamma-producing CD8(+) T cell effectors in the lungs. In CD4(-) mice, depletion of CD8(+) T cells resulted in increased intracellular infection of pulmonary macrophages by C. neoformans, increasing the pulmonary burden of the infection. Neutralization of IFN-gamma in CD4(-)CD8(+) mice similarly increased macrophage infection by C. neoformans, thereby blocking the protection provided by CD8(+) T cells. Altogether, these data support the hypothesis that effector CD8(+) T cell function is independent of CD4(+) T cells and that IFN-gamma production from CD8(+) T cells plays a role in controlling C. neoformans by limiting survival of C. neoformans within macrophages.     

The host environment regulates the function of CD8+ graft-versus-host-reactive effector cells

We have examined how the host environment influences the graft-vs-leukemia (GVL) response following transfer of donor T cells to allogeneic chimeras. Donor T cells induce significant GVL when administered in large numbers to established mixed chimeras (MC). However, when using limiting numbers of T cells, we found that late transfer to MC induced less GVL than did early transfer to freshly irradiated allogeneic recipients. Late donor T cell transfer to MC was associated with marked accumulation of anti-host CD8 cells within the spleen, but delayed kinetics of differentiation, reduced expression of effector molecules including IFN-gamma, impaired cytotoxicity, and higher rates of sustained apoptosis. Furthermore, in contrast to the spleen, we observed a significant delay in donor CD8 cell recruitment to the bone marrow, a key location for hematopoietic tumors. Increasing the numbers of T cells transferred to MC led to the enhancement of CTL activity and detectable increases in absolute numbers of IFN-gamma(+) cells without inducing graft-vs-host disease (GVHD). TLR-induced systemic inflammation accelerated differentiation of functional CTL in MC but was associated with severe GVHD. In the absence of inflammation, both recipient T and non-T cell populations impeded the full development of GVHD-inducing effector function. We conclude that per-cell deficits in the function of donor CD8 cells activated in MC may be overcome by transferring larger numbers of T cells without inducing GVHD.     

A new dynamic model of CD8+ T effector cell responses via CD4+ T helper-antigen-presenting cells

A long-standing paradox in cellular immunology has been the conditional requirement for CD4(+) Th cells in priming of CD8(+) CTL responses. We propose a new dynamic model of CD4(+) Th cells in priming of Th-dependent CD8(+) CTL responses. We demonstrate that OT II CD4(+) T cells activated by OVA-pulsed dendritic cells (DC(OVA)) are Th1 phenotype. They acquire the immune synapse-composed MHC II/OVAII peptide complexes and costimulatory molecules (CD54 and CD80) as well as the bystander MHC class I/OVAI peptide complexes from the DC(OVA) by DC(OVA) stimulation and thus also the potential to act themselves as APCs. These CD4(+) Th-APCs stimulate naive OT I CD8(+) T cell proliferation through signal 1 (MHC I/OVAI/TCR) and signal 2 (e.g., CD54/LFA-1 and CD80/CD28) interactions and IL-2 help. In vivo, they stimulate CD8(+) T cell proliferation and differentiation into CTLs and induce effective OVA-specific antitumor immunity. Taken together, this study demonstrates that CD4(+) Th cells carrying acquired DC Ag-presenting machinery can, by themselves, efficiently stimulate CTL responses. These results have substantial implications for research in antitumor and other aspects of immunity.     

Antibody-independent antiviral function of memory CD4+ T cells in vivo requires regulatory signals from CD8+ effector T cells

Previous studies have shown that vaccine-primed CD4(+) T cells can mediate accelerated clearance of respiratory virus infection. However, the relative contributions of Ab and CD8(+) T cells, and the mechanism of viral clearance, are poorly understood. Here we show that control of a Sendai virus infection by primed CD4(+) T cells is mediated through the production of IFN-gamma and does not depend on Ab. This effect is critically dependent on CD8(+) cells for the expansion of CD4(+) T cells in the lymph nodes and the recruitment of memory CD4(+) T cells to the lungs. Passive transfer of a CD8(+) T cell supernatant into CD8(+) T cell-depleted, hemagglutinin-neuraminidase (HN)(421-436)-immune muMT mice substantially restored the virus-specific memory CD4(+) response and enhanced viral control in the lung. Together, the data demonstrate for the first time that in vivo primed CD4(+) T cells have the capacity to control a respiratory virus infection in the lung by an Ab-independent mechanism, provided that CD8(+) T cell "help" in the form of soluble factor(s) is available during the virus infection. These studies highlight the importance of synergistic interactions between CD4(+) and CD8(+) T cell subsets in the generation of optimal antiviral immunity.     

Disparate cytotoxic activity of nickel-specific CD8+ and CD4+ T cell subsets against keratinocytes

Allergic contact dermatitis (ACD) is the result of an exaggerated immune reaction to haptens mediated by skin-homing T cells, but the effector mechanisms responsible for the tissue damage are poorly understood. Here we studied the capacity of distinct subsets of hapten-specific T cells to induce apoptosis in autologous keratinocytes. Skin- and blood-derived nickel-specific CD8+ T cytotoxic 1 (Tc1) and Tc2 clones as well as CD4+ Th1 and Th2 expressed the cutaneous lymphocyte-associated Ag and exhibited strong MHC-restricted cytotoxicity against nickel-coupled B lymphoblasts, as detected by the [3H]TdR release assay. Both Tc1 and Tc2 clones, but not CD4+ T cells, displayed a significant cytotoxic activity against resting nickel-modified keratinocytes. Following IFN-gamma treatment, keratinocytes expressed MHC class II and ICAM-1 and became susceptible to Th1-mediated, but not Th2-mediated, cytotoxicity. The molecules of the two major cytotoxic pathways, Fas ligand (FasL) and perforin, were expressed by Tc1, Tc2, and Th1 cells, whereas Th2 cells expressed only FasL. Experiments performed in the presence of specific inhibitors of the perforin (concanamycin A) and FasL (brefeldin A) pathway indicated that perforin-mediated killing dominated in Tc1 and Tc2, and FasL-mediated cytotoxicity prevailed in Th2 clones, with a more heterogeneous behavior in the case of Th1 cells. Finally, perforin mRNA was expressed in ACD lesional skin, as assessed by RT-PCR analysis. In aggregate, our results indicate that keratinocytes can be target of multiple hapten-specific CTL responses, that may have distinct roles in the epidermal injury during ACD.     

Cutting edge: cytolytic effector function in human circulating CD8+ T cells closely correlates with CD56 surface expression

Recent data suggest that human effector CD8+ T cells express a distinct CD27-CD45RAhigh (CD57+CD28-CD11ahigh) phenotype. Here, we propose that CTL effector function correlates better with CD56 (neuronal cell adhesion molecule (NCAM)) surface expression. CD56 was absent on cord blood CD8+ T cells, but was expressed by 4-30% of freshly isolated circulating CD8+ T cells from 15 adults. Dramatic oligoclonal expansions in 3/3 individuals were confined to the CD56+ subset of CD8+ T cells. The CD56+ subset generally contained high amounts of intracellular perforin and granzyme B. Finally, direct cytolytic capacity was closely restricted to the CD56+(CD45RAhigh) cells, better than to CD27-CD45RAhigh cells in 5/5 individuals analyzed. Thus, the phenotype corresponding to the circulating effector CD8+ T cell pool may be simplified and more precisely defined by the use of just two surface markers: CD8 and CD56.     

Electroporation enables plasmid vaccines to elicit CD8+ T cell responses in the absence of CD4+ T cells

In vivo electroporation dramatically enhances plasmid vaccine efficacy. This enhancement can be attributed to increased plasmid delivery and, possibly, to some undefined adjuvant properties. Previous reports have demonstrated CD8(+) T cell priming by plasmid vaccines is strongly dependent upon CD4(+) T cell help. Indeed, the efficacy of a plasmid vaccine expressing Escherichia coli beta-galactosidase was severely attenuated in MHC class II-deficient (C2D) mice. To determine whether electroporation could compensate for the absence of CD4(+) T cell help, C2D mice were immunized by a single administration of plasmid in combination with electroporation using two conditions which differed only by the duration of the pulse (20 or 50 msec). Both conditions elicited robust cellular and humoral responses in wild-type mice, as measured by IFN-gamma ELISPOT, anti-beta-galactosidase ELISA, and protection from virus challenge. In C2D mice, the cellular response produced by the vaccine combined with the 50-msec pulse, as measured by ELISPOT, was identical to the response in wild-type mice. The 20-msec pulse elicited a milder response that was approximately one-fifth that of the response elicited by the 50-msec pulse. By contrast, the 20-msec conditions provided comparable protection in both wild-type and C2D recipients whereas the protection elicited by the 50-msec conditions in C2D mice was weaker than in wild-type mice. Further investigation is required to understand the discordance between the ELISPOT results and outcome of virus challenge in the C2D mice. Nonetheless, using this technique to prime CD8(+) T cells using plasmid vaccines may prove extremely useful when immunizing hosts with limiting CD4(+) T cell function, such as AIDS patients.     

4-1BB ligand-mediated costimulation of human T cells induces CD4 and CD8 T cell expansion, cytokine production, and the development of cytolytic effector function

4-1BB (CD137) is a costimulatory member of the TNFR family expressed on activated T cells. Its ligand, 4-1BBL, is expressed on activated APC. In the mouse, CD8 T cells are preferentially activated by agonistic anti-murine 4-1BB Abs. However, murine 4-1BBL can stimulate both CD4 and CD8 T cells. To date, there are only limited data on the effects of 4-1BBL on human T cell responses. To further understand the role of 4-1BBL in human T cell responses, we compared human CD4 and CD8 T cell responses to transfected human 4-1BBL plus TCR-mediated stimulation. Both human CD4 and CD8 T cells responded to 4-1BBL. The presence of 4-1BBL on the APC led to increased expansion, cytokine production, and the development of cytolytic effector function by human T cells. In unfractionated T cell cultures, CD4 and CD8 T cells could expand to a similar extent in response to signals through the TCR and 4-1BB, as measured by CFSE labeling and by quantitating T cell numbers in the cultures. In contrast to the results with total T cells, isolated CD8 T cells produced less IL-2 and expanded to a lesser extent than isolated CD4 T cells responding to 4-1BBL. Thus, 4-1BBL is most effective when both CD4 and CD8 T cells are included in the cultures. CD28 and 4-1BB were found to synergize in the induction of IL-2 by human T cells, and CTLA-Ig partially blocked 4-1BBL-dependent IL-2 production. However, a portion of the 4-1BBL-mediated effects were independent of CD28-B7 interaction.     

Regulation of CD8+ T cells undergoing primary and secondary responses to infection in the same host

Naive Ag-specific CD8(+) T cells expand, contract, and become memory cells after infection and/or vaccination. Memory CD8(+) T cells provide faster, more effective secondary responses against repeated exposure to the same pathogen. Using an adoptive transfer system with low numbers of trackable nontransgenic memory CD8(+) T cells, we showed that secondary responses can be comprised of both primary (naive) and secondary (memory) CD8(+) T cells after bacterial (Listeria monocytogenes) and/or viral (lymphocytic choriomeningitis virus) infections. The level of memory CD8(+) T cells present at the time of infection inversely correlated with the magnitude of primary CD8(+) T cell responses against the same epitope but directly correlated with the level of protection against infection. However, similar numbers of Ag-specific CD8(+) T cells were found 8 days postinfection no matter how many memory cells were present at the time of infection. Rapid contraction of primary CD8(+) T cell responses was not influenced by the presence of memory CD8(+) T cells. However, contraction of secondary CD8(+) T cell responses was markedly prolonged compared with primary responses in the same host mice. This situation occurred in response to lymphocytic choriomeningitis virus or L. monocytogenes infection and for CD8(+) T cell responses against multiple epitopes. The delayed contraction of secondary CD8(+) T cells was also observed after immunization with peptide-coated dendritic cells. Together, the results show that the level of memory CD8(+) T cells influences protective immunity and activation of naive precursors specific for the same epitope but has little impact on the magnitude or program of the CD8(+) T cell response.     

Progressive differentiation and commitment of CD8+ T cells to a poorly cytolytic CD8low phenotype in the presence of IL-4

Exposure to IL-4 during activation of naive murine CD8+ T cells leads to generation of IL-4-producing effector cells with reduced surface CD8, low perforin, granzyme B and granzyme C mRNA, and poor cytolytic function. We show in this study that maximal development of these cells depended on exposure to IL-4 for the first 5 days of activation. Although IL-4 was not required at later times, CD8 T cell clones continued to lose surface CD8 expression with prolonged culture, suggesting commitment to the CD8low phenotype. This state was reversible in early differentiation. When single CD8low cells from 4-day cultures were cultured without IL-4, 65% gave rise to clones that partly or wholly comprised CD8high cells; the proportion of reverted clones was reduced or increased when the cells were cloned in the presence of IL-4 or anti-IL-4 Ab, respectively. CD8 expression positively correlated with perforin and granzyme A, B, and C mRNA, and negatively correlated with IL-4 mRNA levels among these clones. By contrast, most CD8low cells isolated at later time points maintained their phenotype, produced IL-4, and exhibited poor cytolytic function after many weeks in the absence of exogenous IL-4. We conclude that IL-4-dependent down-regulation of CD8 is associated with progressive differentiation and commitment to yield IL-4-producing cells with little cytolytic activity. These data suggest that the CD4-CD8- cells identified in some disease states may be the product of a previously unrecognized pathway of effector differentiation from conventional CD8+ T cells.     

Subpopulations of CD8+ cytotoxic T cell precursors collaborate in the absence of conventional CD4+ helper T cells.

Four different subpopulations (Ly6Cneg, Ly6Clow, Ly6Cint, and Ly6Chi) of CD8+ T cells were arbitrarily defined on the basis of differential expression of Ly6C Ag. By combining the processes of electronic cell sorting and automated cell deposition, small numbers of respective CD8+ T cell subpopulations were directly deposited into tissue culture wells in which mitogen-stimulated responses were studied. Anti-CD3-stimulated proliferation and IL-2 production were the strongest by Ly6Cneg/Ly6Clow T cells, moderate for Ly6Cint T cells, and highly deficient for Ly6Chi T cells. The level of IL-2 production for Ly6Cneg CD8+ T cells was comparable to that of conventional CD4+ Th cells. Allogeneic stimulator cells elicited a strong cytotoxic response by Ly6Cneg + low but not Ly6Chi CD8+ T cells in the absence of added lymphokines. When IL-2 was supplied in excess, anti-CD3 induced comparable levels of cell proliferation and cytotoxic activity in Ly6Cneg, Ly6Clow, Ly6Cint, and Ly6Chi CD8+ T cells whereas alloantigen stimulated an approximate fivefold higher cytotoxic response by Ly6Chi than Ly6Cneg + low CD8+ T cells. Stimulation of co-cultures of B10 (CD8b) Ly6Cneg + low and congenic B10.CD8a Ly6Chi CD8+ T cells in the absence of added lymphokines, followed by selective elimination of activated CD8.1+ (CD8.2+) T cells by anti-CD8.1 (anti-CD8.2) + C treatment, allowed the demonstration that help provided by Ly6Cneg + low T cells can be effectively used by both Ly6Cneg + low and Ly6Chi T cells in anti-CD3 and alloantigen induced proliferative and cytotoxic responses, respectively.     

Cross-primed CD8+ cytotoxic T cells induce severe Helicobacter-associated gastritis in the absence of CD4+ T cells

BACKGROUND: Although previous studies have reported important roles of CD4(+) type 1-helper T cells and regulatory T cells in Helicobacter-associated gastritis, the significance of CD8(+) cytotoxic T cells remains unknown. To study the roles of CD8(+) T cells, we examined the immune response in the gastric mucosa of Helicobacter felis-infected major histocompatibility complex (MHC) class II-deficient (II(-/-)) mice, which lack CD4(+) T cells. MATERIALS AND METHODS: Stomachs from H. felis-infected wild-type and infected MHC II(-/-) mice were examined histologically and immunohistochemically. Gastric acidity and serum levels of anti-H. felis antibodies were measured. The expression of pro-inflammatory and anti-inflammatory cytokine, Fas-ligand, perforin, and Foxp3 genes in the gastric mucosa was investigated. RESULTS: H. felis-infected MHC II(-/-) mice developed severe gastritis, accompanied by marked infiltration of CD8(+) cells. At 1 and 2 months after inoculation, mucosal inflammation and atrophy were more severe in MHC II(-/-) mice, although gastritis had reached similar advanced stages at 3 months after inoculation. There was little infiltration of CD4(+) cells, and no Foxp3-positive cells were detected in the gastric mucosa of the infected MHC II(-/-) mice. The expression of the interleukin-1beta and Fas-ligand genes was up regulated, but that of Foxp3 was down regulated in the infected MHC II(-/-) mice. Serum levels of anti-H. felis antibodies were lower in the infected MHC II(-/-) mice, despite severe gastritis. CONCLUSIONS: The present study suggests that cross-primed CD8(+) cytotoxic T cells can induce severe H.-associated gastritis in the absence of CD4(+) helper T cells and that Foxp3-positive cells may have an important role in the control of gastric inflammation.     

NK cells regulate CD8+ T cell effector function in response to an intracellular pathogen

We studied the role of NK cells in regulating human CD8+ T cell effector function against mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Depletion of NK cells from PBMC of healthy tuberculin reactors reduced the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ cells and decreased their capacity to lyse M. tuberculosis-infected monocytes. The frequency of CD8+ IFN-gamma+ cells was restored by soluble factors produced by activated NK cells and was dependent on IFN-gamma, IL-15, and IL-18. M. tuberculosis-activated NK cells produced IFN-gamma, activated NK cells stimulated infected monocytes to produce IL-15 and IL-18, and production of IL-15 and IL-18 were inhibited by anti-IFN-gamma. These findings suggest that NK cells maintain the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ T cells by producing IFN-gamma, which elicits secretion of IL-15 and IL-18 by monocytes. These monokines in turn favor expansion of Tc1 CD8+ T cells. The capacity of NK cells to prime CD8+ T cells to lyse M. tuberculosis-infected target cells required cell-cell contact between NK cells and infected monocytes and depended on interactions between the CD40 ligand on NK cells and CD40 on infected monocytes. NK cells link the innate and the adaptive immune responses by optimizing the capacity of CD8+ T cells to produce IFN-gamma and to lyse infected cells, functions that are critical for protective immunity against M. tuberculosis and other intracellular pathogens.     

Three memory subsets of human CD8+ T cells differently expressing three cytolytic effector molecules

Multicolor flow cytometric analysis for the expression of three effector molecules, i.e., perforin (Per), granzyme A (GraA), and granzyme B (GraB), in human CD8(+) T cells demonstrated that they included five subpopulations, implying the following pathway for the differentiation of CD8(+) T cells: Per(-)GraA(-)GraB(-)-->Per(-)GraA(+)GraB(-)-->Per(low)GraA(+)GraB(-)--> Per(low)GraA(+)GraB(+)-->Per(high)GraA(+)GraB(+). The analysis of the expression of these molecules in the subsets classified by the combination of the expression of CCR7 and CD45RA or by that of CD27, CD28, and CD45RA showed that functional CD8(+) T cell subsets could be partially identified by these phenotypic classifications. However, the functional subsets could be precisely identified by the classification using five cell surface markers or three cell surface markers and three cytolytic molecules. Per(-)GraA(-)GraB(-) and Per(-/low)GraA(+)GraB(-) cells were predominantly found in CCR5(-)CCR7(+) and CCR5(high/low)CCR7(-) subsets, respectively, of CD8(+) T cells expressing the CD27(+)CD28(+)CD45RA(-) phenotype, whereas Per(low)GraA(+)GraB(+) cells were found in the CCR5(low)CCR7(-) subset of those expressing this phenotype and in a part of the CCR5(-/low)CCR7(-) subset of those expressing the CD27(-/low)CD28(-)CD45RA(-/+) phenotype. Ex vivo EBV-specific CD8(+) T cells, which were Per(low/-)GraA(+)GraB(-/+) cells, hardly or very weakly killed the target cells, indicating that these were not effector T cells. These findings suggest that the Per(-)GraA(-)GraB(-), Per(-/low)GraA(+)GraB(-), and Per(low)GraA(+)GraB(+) cells were central memory, early effector memory, and late effector memory T cells, respectively. Per(-/low)GraA(+)GraB(-) cells gained GraB expression after TCR stimulation, indicating that early effector memory T cells could differentiate into late effector and effector T cells. The present study showed the existence of three memory subsets and the pathway for their differentiation.     

Anti-CD8 antibodies can trigger CD8+ T cell effector function in the absence of TCR engagement and improve peptide-MHCI tetramer staining

CD8(+) T cells recognize immunogenic peptides presented at the cell surface bound to MHCI molecules. Ag recognition involves the binding of both TCR and CD8 coreceptor to the same peptide-MHCI (pMHCI) ligand. Specificity is determined by the TCR, whereas CD8 mediates effects on Ag sensitivity. Anti-CD8 Abs have been used extensively to examine the role of CD8 in CD8(+) T cell activation. However, as previous studies have yielded conflicting results, it is unclear from the literature whether anti-CD8 Abs per se are capable of inducing effector function. In this article, we report on the ability of seven monoclonal anti-human CD8 Abs to activate six human CD8(+) T cell clones with a total of five different specificities. Six of seven anti-human CD8 Abs tested did not activate CD8(+) T cells. In contrast, one anti-human CD8 Ab, OKT8, induced effector function in all CD8(+) T cells examined. Moreover, OKT8 was found to enhance TCR/pMHCI on-rates and, as a consequence, could be used to improve pMHCI tetramer staining and the visualization of Ag-specific CD8(+) T cells. The anti-mouse CD8 Abs, CT-CD8a and CT-CD8b, also activated CD8(+) T cells despite opposing effects on pMHCI tetramer staining. The observed heterogeneity in the ability of anti-CD8 Abs to trigger T cell effector function provides an explanation for the apparent incongruity observed in previous studies and should be taken into consideration when interpreting results generated with these reagents. Furthermore, the ability of Ab-mediated CD8 engagement to deliver an activation signal underscores the importance of CD8 in CD8(+) T cell signaling.     

Superantigen-induced regulatory T cells display different suppressive functions in the presence or absence of natural CD4+CD25+ regulatory T cells in vivo

Repeated exposures to both microbial and innocuous Ags in vivo have been reported to both eliminate and tolerize T cells after their initial activation and expansion. The remaining tolerant T cells have been shown to suppress the response of naive T cells in vitro. This feature is reminiscent of natural CD4(+)CD25(+) regulatory T cells. However, it is not known whether the regulatory function of in vivo-tolerized T cells is similar to the function of natural CD4(+)CD25(+) regulatory T cells. In this study, we demonstrate that CD4(+)CD25(+) as well as CD4(+)CD25(-) T cells isolated from mice treated with superantigen three consecutive times to induce tolerance were functionally comparable to natural CD4(+)CD25(+) regulatory T cells, albeit more potent. The different subpopulations of in vivo-tolerized CD4(+) T cells efficiently down-modulated costimulatory molecules on dendritic cells, and their suppressive functions were strictly cell contact dependent. Importantly, we demonstrate that conventional CD4(+)CD25(-) T cells could also be induced to acquire regulatory functions by the same regimen in the absence of natural regulatory T cells in vivo, but that such regulatory cells were functionally different.     

Nucleocapsid or spike protein-specific CD4+ T lymphocytes protect against coronavirus-induced encephalomyelitis in the absence of CD8+ T cells.

To investigate the antiviral CD4+ T cell response in coronavirus MHV-JHM-induced encephalomyelitis, spleen and thymic lymphocytes from diseased rats were stimulated in culture with virus Ag, expanded and tested for their specificity to viral proteins and nucleocapsid (N) and spike (S) proteins that had been expressed in bacteria. A strong T cell response specific for N was measurable during acute disease, whereas S-specific T cells were only detectable in rats with a later onset of disease. CD4+ T cell lines with specificity for virus and either N or S protein were established and their influence on the course of a mouse hepatitis virus-JHM infection was investigated. All lines were of the CD4+ phenotype. Both N and S protein-specific CD4+ T cells conferred protection to infected Lewis rats and reduced the amount of infectious virus in the central nervous system. After transfer of CD4+ T cells and challenge with virus, an increase in the antiviral IgM response occurred, but neutralizing antibodies were not detectable during the period of virus clearance. Previous CD8+ cell depletion did not abrogate protection mediated by CD4+ T cell line transfer.     

Induction of CD70 on dendritic cells through CD40 or TLR stimulation contributes to the development of CD8+ T cell responses in the absence of CD4+ T cells

The expansion of CD8(+) T cells in response to Ag can be characterized as either dependent or independent of CD4(+) T cells. The factors that influence this dichotomy are poorly understood but may be dependent upon the degree of inflammation associated with the Ag. Using dendritic cells derived from MHC class II-deficient mice to avoid interaction with CD4(+) T cells in vivo, we have compared the immunogenicity of peptide-pulsed dendritic cells stimulated with molecules associated with infection to those stimulated via CD40. In the absence of CD4(+) T cell help, the expansion of primary CD8(+) T cells after immunization with TNF-alpha- or poly(I:C)-stimulated dendritic cells was minimal. In comparison, LPS- or CpG-stimulated dendritic cells elicited substantial primary CD8(+) T cell responses, though not to the same magnitude generated by immunization with CD40L-stimulated dendritic cells. Remarkably, mice immunized with any stimulated dendritic cell population generated fully functional recall CD8(+) T cells without the aid of CD4(+) T cell help. The observed hierarchy of immunogenicity was closely correlated with the expression of CD70 (CD27L) on the stimulated dendritic cells, and Ab-mediated blockade of CD70 substantially prevented the CD4(+) T cell-independent expansion of primary CD8(+) T cells. These results indicate that the expression of CD70 on dendritic cells is an important determinant for helper-dependence of primary CD8(+) T cell expansion and provide an explanation for the ability of a variety of pathogens to stimulate primary CD8(+) T cell responses in the absence of CD4(+) T cells.     

In vivo CD86 blockade inhibits CD4+ T cell activation, whereas CD80 blockade potentiates CD8+ T cell activation and CTL effector function

To address whether a functional dichotomy exists between CD80 and CD86 in naive T cell activation in vivo, we administered anti-CD80 or CD86 blocking mAb alone or in combination to mice with parent-into-F(1) graft-vs-host disease (GVHD). In this model, the injection of naive parental T cells into unirradiated F(1) mice results in either a Th1 cytokine-driven, cell-mediated immune response (acute GVHD) or a Th2 cytokine-driven, Ab-mediated response (chronic GVHD) in the same F(1) recipient. Combined CD80/CD86 blockade beginning at the time of donor cell transfer mimicked previous results seen with CTLA4Ig and completely abrogated either acute or chronic GVHD by preventing the activation and maturation of donor CD4(+) T cells as measured by a block in acquisition of memory marker phenotype and cytokine production. Similar results were seen with selective CD86 blockade; however, the degree of CD4 inhibition was always less than that seen with combined CD80/CD86 blockade. A more striking effect was seen with selective CD80 blockade in that chronic GVHD was converted to acute GVHD. This effect was associated with the induction of Th1 cytokine production, donor CD8(+) T cell activation, and development of antihost CTL. The similarity of this effect to that reported for selective CTLA4 blockade suggests that CD80 is a critical ligand for CTLA4 in mediating the down-regulation of Th1 responses and CD8(+) T cell activation. In contrast, CD86 is critical for the activation of naive CD4(+) T cells in either a Th1 or a Th2 cytokine-mediated response.     

Selective bystander proliferation of memory CD4+ and CD8+ T cells upon NK T or T cell activation

Ag-experienced or memory T cells have increased reactivity to recall Ag, and can be distinguished from naive T cells by altered expression of surface markers such as CD44. Memory T cells have a high turnover rate, and CD8(+) memory T cells proliferate upon viral infection, in the presence of IFN-alphabeta and/or IL-15. In this study, we extend these findings by showing that activated NKT cells and superantigen-activated T cells induce extensive bystander proliferation of both CD8(+) and CD4(+) memory T cells. Moreover, proliferation of memory T cells can be induced by an IFN-alphabeta-independent, but IFN-gamma- or IL-12-dependent pathway. In these conditions of bystander activation, proliferating memory (CD44(high)) T cells do not derive from activation of naive (CD44(low)) T cells, but rather from bona fide memory CD44(high) T cells. Together, these data demonstrate that distinct pathways can induce bystander proliferation of memory T cells.